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NUAK2 Amplification Coupled with PTEN Deficiency Promote Melanoma Development Via CDK Activation

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NUAK2 Amplification Coupled with PTEN Deficiency Promote Melanoma Development Via CDK Activation Author Manuscript Published OnlineFirst on April 1, 2015; DOI: 10.1158/0008-5472.CAN-13-3209 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Namiki et al. - 1 NUAK2 amplification coupled with PTEN deficiency promote melanoma development via CDK activation Takeshi Namiki1,2,6, Tomonori Yaguchi2, Kenta Nakamura2,3, Julio C. Valencia1, Sergio G. Coelho1, Lanlan Yin1, Masakazu Kawaguchi1, Wilfred D. Vieira1, Yasuhiko Kaneko4, Atsushi Tanemura5, Ichiro Katayama5, Hiroo Yokozeki6, Yutaka Kawakami2 & Vincent J. Hearing1 1Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20814, USA 2Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo 160-8582, Japan 3Department of Dermatology, Shinshu University School of Medicine, Matsumoto-shi, Nagano 390-8621, Japan 4Research Institute for Clinical Oncology, Saitama Cancer Center, Kitaadachi, Saitama 362-0806, Japan 5Department of Dermatology, Osaka University Graduate School of Medicine, Suita-shi, Osaka 565-0871, Japan 6Department of Dermatology, Tokyo Medical and Dental University Graduate School and Faculty of Medicine, Bunkyo-ku, Tokyo 113-8519, Japan Running Title: Inhibition of CDK2 inhibits melanoma growth Total Words: 3,174 words (Max: 5,000 words) Number of Figures / Tables: 6 Figures, 1 Table KeyWords: NUAK2; PTEN; PI3K pathway; CDK2; cell cycle; melanoma Conflicts of Interest: The authors have declared that no conflict of interest exists. Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 1, 2015; DOI: 10.1158/0008-5472.CAN-13-3209 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Namiki et al. - 2 Address correspondence to: Dr. Yutaka Kawakami, Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan; Tel.: 81-3-5363-3778; FAX: 81-3-5362-9259; E-mail: [email protected] Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 1, 2015; DOI: 10.1158/0008-5472.CAN-13-3209 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Namiki et al. - 3 Abstract (194 words, limit = 250) The AMPK-related kinase NUAK2 has been implicated in melanoma growth and survival outcomes but its therapeutic utility has yet to be confirmed. In this study, we show how its genetic amplication in PTEN-deficient melanomas may rationalize the use of CDK2 inhibitors as a therapeutic strategy. Analysis of array-CGH data revealed that PTEN deficiency is coupled tightly with genomic amplification encompassing the NUAK2 locus, a finding strengthened by immunohistochemical evidence that phospho-Akt overexpression was correlated with NUAK2 expression in clinical specimens of acral melanoma. Functional studies in melanoma cells showed that inactivation of the PI3K pathway upregulated p21 expression and reduced the number of cells in S phase. NUAK2 silencing and inactivation of the PI3K pathway efficiently controlled CDK2 expression, whereas CDK2 inactivaiton specifically abrogated the growth of NUAK2-amplified and PTEN-deficient melanoma cells. Immunohistochemical analyses confirmed an association of CDK2 expression with NUAK2 amplification and p-Akt expression in melanomas. Lastly, pharmacological inhibition of CDK2 was sufficient to suppress the growth of NUAK2-amplified and PTEN-deficient melanoma cells in vitro and in vivo. Overall, our results identify show how CDK2 blockade may offer a promising therapy for genetically-defined melanomas where NUAK2 is amplified and PTEN is deleted. Precis This study defines a novel gene activation event that is critical with PTEN deletion for progression of cutaneous melanomas, with implications that rationalize the use of CDK2 inhibitors to treat melanomas with those genomic aberrations Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 1, 2015; DOI: 10.1158/0008-5472.CAN-13-3209 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Namiki et al. - 4 Introduction Recent advances in cancer genomics facilitate the elucidation of aberrant downstream pathways in tumor cells with genomic aberrations and pave the way to develop specific therapies for novel oncogenes and tumor-suppressor genes in many types of cancers (1-4). In melanomas, several genomic aberrations, such as mutations, amplifications and deletions in BRAF, NRAS, INK4A, MITF, PREX2, GNAQ and KIT, have been reported, and recent analyses using array-CGH data also suggested that NUAK2, which resides at chromosome 1q32, is an important gene that regulates cell cycle progression and cell migration in melanoma cells (2;5-14). The significance of NUAK2 in melanomagenesis is highlighted by the fact that high expression of NUAK2 has an impact on the survival of patients with acral melanomas in addition to the fact that NUAK2 participates in the regulation of cell proliferation of melanomas in general (12). On the other hand, synergistic effects of several genomic aberrations are also quite important to facilitate tumorigenesis of cancer cells such as that the PI3K pathway participates in melanomagenesis (15;16). The elucidation of genomic aberrations including mutations has progressed using systematic approaches (17). However, detailed mechanisms controlling cell cycle progression by NUAK2 and additional genes remain to be elucidated. Analyses of cell cycle progression in NRAS-mutated and MITF-amplified melanomas showed that control of the cell cycle is differently regulated by CDKs in melanoma cells, where CDK4 is a key driver in NRAS-mutant melanomas, while CDK2 has a pivotal role in melanomas with high expression of MITF (18-20) Those results imply that elucidation of mechanisms regulating the cell cycle by different genomic aberrations should reveal the different impact of CDKs on the cell cycle. In melanomas, BRAF mutations have been identified as activating mutations that facilitate Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 1, 2015; DOI: 10.1158/0008-5472.CAN-13-3209 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Namiki et al. - 5 melanomagenesis, and this discovery accelerated molecular targeted therapies against melanomas using drugs such as vemurafenib and dabrafenib (1;2). However BRAF mutations have diverse discrepancies among subtypes of melanomas (21). Some subtypes of melanomas, such as acral and mucosal melanomas, have low frequencies of BRAF mutations and are speculated to respond poorly to those therapies targeting BRAF mutations. Molecular targeted therapies aimed at genomic aberrations other than BRAF mutations should be developed for better management of patients with those subtypes of melanomas. In this study, we explore additional genomic aberrations and downstream pathways of NUAK2, and demonstrate that NUAK2 and the PI3K pathway coordinately control CDK2. In addition, we showed that CDK2 is an efficient therapeutic target by abrogating the growth of cutaneous melanomas. Material and Methods Tumor specimens We obtained 91 paraffin-embedded specimens of primary melanomas from 3 Institutions. This study was approved by the Tokyo Medical and Dental University Research Committee, the Osaka University Clinical Research Committee and the Saitama Cancer Center Research Ethics Committee. Fifty-six tumors were classified as acral melanomas and 35 as non-chronic sun-induced damage (CSD) melanomas, but none was a CSD melanoma according to the definition by Curtin and colleagues (22). Cell lines Normal human melanocyte and melanoma cell lines were cultured and maintained as previously described (23). C32, A375 and Malme-3M melanoma cells were purchased from the Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2015 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 1, 2015; DOI: 10.1158/0008-5472.CAN-13-3209 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Namiki et al. - 6 American Type Culture Collection (Manassas, VA). SKMel28 and SKMel23 melanoma cells were kindly provided by the Surgery Branch, NCI/NIH (Bethesda, MD). SM2-1 melanoma cells were kindly provided by Dr. H. Murata (Shinshu University, Matsumoto, Japan). The Mel2 melanoma cell line was established from a lymph node metastasis of a 68-year-old Japanese male acral melanoma patient in 1998 and the mel18 melanoma cell line was established from a lymph node metastasis of a 51-year-old Japanese male acral melanoma patient in 1998 in our laboratory, as described previously (24). C32, mel2, mel18 and SM2-1 melanoma cell lines were cultured in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin at 37ºC in a 5% CO2 incubator. All other melanoma cells were cultured in DMEM with 5% FBS. The original C32 , A375 and Malme-3M melanoma cells were STR DNA profiled in 2012. Vectors, siRNA transfection
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